Gram-negative disease and its most serious complications, e.g., bacteremia and endotoxemia, are the cause of significant morbidity and mortality in human patients. This is particularly true of the gram-negative organism Pseudomonas aeruginosa, which has been increasingly associated with bacterial infections, especially nosocomial infections, over the last fifty years.
During the past few decades, antibiotics have been the therapy of choice for controlling gram-negative disease. The continued high morbidity and high mortality associated with gram-negative bacterial disease, however, is indicative of the limitations of antibiotic therapy, particularly with respect to P. aeruginosa. (See, for example, Andriole, V. G., "Pseudomonas Bacteremia: Can Antibiotic Therapy Improve Survival?", J. Lab. Clin. Med. [1978] 94:196-199). This has prompted the search for alternative methods of prevention and treatment.
One method that has been considered is augmentation of the host's immune system by active or passive immunization. For instance, it has been observed that active immunization of humans or experimental animals with whole cell bacterial vaccines or purified bacterial endotoxins from P. aeruginosa leads to the development of specific opsonic antibodies directed primarily against determinants on the repeating oligosaccharide units of the lipopolysaccharide (LPS) molecules located on the outer cell membrane of P. aeruginosa (see Pollack, M., Immunoglobulins: Characteristics and Uses of Intravenous Preparations, Alving, B. M., and Finlayson, J. S., eds., pp. 73-79, U.S. Department of Health and Human Services, 1979). Such antibodies, whether actively engendered or passively transferred, have been shown to be protective against the lethal effects of P. aeruginosa infection in a variety of animal models (Pollack, supra) and in some preliminary investigations with humans (see Young, L. S. and Pollack, M., Pseudomonas aeruginosa, Sabath, L., ed., pp. 119-132, Hans Huber, 1980).
The above reports suggest that immunotherapeutic approaches could be utilized to prevent and treat bacterial disease due to P. aeruginosa, such as by administering pooled human immune globulins that contain antibodies against the infecting strain(s). Human immune globulins are defined herein as that portion of fractionated human plasma that is enriched for antibodies, among which are represented specific antibodies to strains of P. aeruginosa. Due to certain inherent limitations in using human immune globulin components, this approach to treatment of disease due to P. aeruginosa remains under investigation (see, for example, Collins, M. S. and Roby, R. E., Am. J. Med., 76(3A):168-174, [1984]), and as yet there are no commercial products available utilizing these components.
One such limitation associated with immune globulin compositions is that they consist of pools of samples from a thousand or more donors, such samples having been preselected for the presence of particular anti-Pseudomonas antibodies. This pooling leads to an averaging of individual antibody titers which, at best, results in modest increases in the resultant titer of the desired antibodies.
Another limitation is that the preselection process itself requires expensive, continuous screening of the donor pool to assure product consistency. Despite these efforts, the immune globulin products can still have considerable variability from batch to batch and among products from different geographic regions.
Yet another such limitation inherent in immune globulin compositions is that their use results in the coincident administration of large quantities of extraneous proteinaceous substances (which may include viruses, such as those recently shown to be associated with Acquired Immune Deficiency Syndrome, or AIDS), having the potential to cause adverse biologic effects. The combination of low titers of desired antibodies and high content of extraneous substances may often limit, to suboptimal levels, the amount of specific and thus beneficial immune globulin(s) administrable to the patient.
In 1975 Kohler and Milstein reported their seminal discovery that certain mouse cell lines could be fused with mouse spleen cells to create hybridomas each of which which would secrete antibodies of a single specificity, i.e., monoclonal antibodies (Kohler, G., and Milstein, C., Nature, 256:495-497 [1975]). With the advent of this technology it became possible, in some cases, to produce large quantities of exquisitely specific murine antibodies to a particular determinant or determinants on antigens. Subsequently, using later-developed technologies, it became possible to produce human monoclonal antibodies (see, e.g., U.S. Pat. No. 4,464,465, which is incorporated herein by reference).
It is recognized that in some situations mouse monoclonal antibodies or compositions of such antibodies may present problems for use in humans. For example, it has been reported that mouse monoclonal antibodies used in trial studies for the treatment of certain human disease can elicit an immune response that renders them noneffective (Levy, R. L., and Miller, R. A., Ann. Rev. Med., 34:107-116 [1983]). However, with recent advances in recombinant DNA technology, such as the production of chimeric mouse/human monoclonal antibodies, these problems may be abated. Also, methods for the production of human monoclonal antibodies are now available (see, Human Hybridomas and Monoclonal Antibodies, Engleman, E. G., et al., eds., Plenum Publishing Corp. [1985], which is incorporated herein by reference).
Using hybridoma and/or cell transformation technology, a number of groups have reported the production of monoclonal antibodies protective against P. aeruginosa infections. Monoclonal antibodies have been produced that are reactive with various epitopes of P. aeruginosa, including single and multi-serotype specific surface epitopes, such as those found in LPS molecules of the bacteria (see, for example, commonly assigned pending U.S. patent application Ser. Nos. 734,624 and 807,394, which are both incorporated herein by reference). Also, protective monoclonal antibodies specific for P. aeruginosa exotoxin A have been produced (see, for example, commonly assigned U.S. patent application Ser. No. 742,170, which is incorporated herein by reference).
While utilizing monoclonal antibodies specific for the LPS region of P. aeruginosa, or the bacteria's exotoxins, may provide sufficient protection in some situations, generally it is preferable to have broader protection capability. For example, in prophylactic treatments for potential infections in humans, it would be preferable to administer an antibody or antibodies protective against a plurality of P. aeruginosa strains. Similarly, in therapeutic applications where the serotype(s) of the infecting strain(s) is not know, it would be preferable to administer an antibody or combination of antibodies effective against most, if not all, of the clinically important P. aeruginosa serotypes, ideally by providing antibodies reactive across traditional serotyping schemes.
One aspect of P. aeruginosa physiology that has been shown to contribute to the organism's virulence is motility, a capability resulting primarily from the presence of a flagellum (see, Montie, T., et al. [1982], Infect. and Immun., 38:1296-1298). P. aeruginosa is characterized by having a single flagellum at one end of its rod-shaped structure. Burned mouse model studies have shown that a greater percentage of mice survived when non-motile P. aeruginosa strains were inoculated into experimental burns than if motile strains were utilized. (McManus, A., et al. [1980], Burns, 6:235-239 and Montie, T., et al. [1982], Infect. and Immun., 38:1296-1298). Other studies on the pathogenesis of P. aeruginosa have alleged that animals immunized with flagella antigen preparations were protected when burned and infected with motile strains of the bacteria (see, Holder, I., et al. [1982], Infect. and Immun., 35:276-280).
Importantly, P. aeruginosa flagella have been studied by serological methods and have been reported to fall into two major antigenic groups designated H1 and H2 by B. Lanyi (1970, Acta Microbiol. Acad. Sci. Hung., 17:35-48) and type a and type b by Ansorg, R. (1978, Zbl. Bakt. Hyg., I. Abt. Orig. A, 242:228-238). Serological typing of flagella by both laboratories showed that H1 flagella (Lanyi, B., supra) or flagella type b (Ansorg, R., supra) was serologically uniform, i.e., no subgroups have been identified. This serologically uniform flagellar type will be referred to as type b. The other major antigen, H2 (Lanyi, B., supra) or type a flagella (Ansorg, R., supra) contained five subgroups. This antigen will be referred to as flagella type a, and the five subgroups as a.sub.0, a.sub.1, a.sub.2, a.sub.3, and a.sub.4. The five subgroups of type a are expressed in varying combinations on different strains of type a bearing P. aeruginosa with the exception of the antigen a.sub.0. The a.sub.0 antigen was found on all type a flagella, although the degree to which it was expressed varied among strains.
A serotyping scheme based on the heat stable major somatic antigens of P. aeruginosa is referred to as the Habs scheme, which has recently been incorporated into the International Antigenic Typing System scheme. (See, Liu, Int. J. Syst. Bacteriol., 33:256 [1983].) The flagella types of P. aeruginosa Habs reference strains have been characterized by immunofluorescence with polyclonal sera by R. Ansorg (1978, Zbl. Bakt. Hyg., I. Abt. Orig. A, 242:228-238) or by slide coagglutination (Ansorg, R., et al., 1984, J. Clin. Microbiol., 20:84-88). Habs strains 2, 3, 4, 5, 7, 10, 11, and 12 are flagella type b bearing strains, and Habs strains 1, 6, 8, and 9 bear type a flagella. Thus, a large number of strains of P. aeruginosa could possibly be recognized by a small number of monoclonal antibodies specific for flagellar proteins.
Accordingly, there exists a significant need for monoclonal antibodies capable of reacting with epitopes on flagellar proteins and, in some cases, also providing protection against multiple serotypes of P. aeruginosa. Further, some of these antibodies should be suitable for use as prophylactic and therapeutic treatments of P. aeruginosa infections, as well as the diagnosis of such infections. The present invention fulfills these needs.